isolating large plasmids - is it possible (Aug/26/2006 )
one of my friends has isolated a strain of streptomyces whichs is tolerant to cadmium.
now the problem is she is not able to isolate the plasmid for it by conventional methods.
I read somewhere that this strain has large plasmids and if it is so how can they be separated?
Why do you think this is on a plasmid -- it could easily be a genomic construct, such as an insertion element or integrated phage, or even a mutation of a normally present gene. The species might even be naturally resistant.
Could she lyse the bacteria while embedded in the gel and run it to see what she will get?
Try both a plasmid prep and a genomic prep. You'll have both fractions to try to amplify, sequence or subclone the gene(s) involved. Commercial kits or traditional methods are available for both purification techniques.
thanks for the replies
to answer few questions,
phage 434 she thinks that this is a plasmid since metal resistance is generally believed to be on plasmid. it will be very nice of you if you could name an organism whose metal reistance gene is in the genomic DNA. I could find more about it and tell her.
I have question genehunter how does one lyse bacteria embedded in gel?
I think you will this information helpful in solving the problem that the genome of this species is not sequenced and the large plasmids of 330kb are reported from this species. and when she does plasmid all she gets is a smear.
She can make gel plug as described in http://jcm.asm.org/cgi/content/abstract/34/10/2598
i've used this method for bacmid preparation (roughly 135kpb DNA)
2ml culture with appropriate selection
do not vortex, pipett always gently, do not freeze thaw (but 2cylces don't affect prep quality)
put in eppi. Spin 1' 13000g
resuspend cells with 0,3 ml solution 50mM Tris HCL pH :8,0 ; 10mM EDTA ; 100 µg/ml RNase (P1 prep quiagen)
add 0,3 ml d’une solution 0,2 N NaOH, 1%SDS, homogenize gently.
Let incubate 5-10' RT
add 0,3 ml of solution Na acétate 3M homogenize gently during addition.5-10' on ice
spin 10 min 13 000g R.T.
Put 0.8ml IprOH and add the supernatant of the spinhomogenize slow by inverting tube.
5-10' on ice and spin 15' RT 13000g
wash by 0.5ml ethanol 70% twice
13 – Centrifuger 5 min à 13 000g
Let dry 5 10 min R.T
Dissolve in 40 µl TE.
CONSERVER LES BACMIDES A 4°C (ne pas congeler)
Fred: isn't this essentially a genomic DNA prep? I don't see where the genomic DNA is removed in this protocol. Of course, this may be OK for many applications.
well, the alkaline lysis step does remove most if not all the chromosomal DNA.
but with a size in ~300kb range that could drastically reduce the yield by this method. Maybe non-ionic detergent lysis method followed by 100,000 g 1hr spin to remove the chromosomal DNA gel could help getting better yield for this giant plasmid.
well i agree with genehunter in the fact the lysis stepremove chromosomal DNA.
May a cesium chloride gradient be applicable here?